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A comparative study of the -subdomains of bovine and human -lactalbumin reveals key differences that correlate with molten globule stability

¹ Department of Chemistry, ² Graduate Program in Biochemistry and Structural Biology, and ³ Graduate Program in Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA

Reprint requests to: Daniel Raleigh, Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA; e-mail: draleigh{at}notes.cc.sunysb.edu; fax: (631) 632-7960.

(RECEIVED July 7, 2004; FINAL REVISION September 6, 2004; ACCEPTED September 13, 2004)

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
The -lactalbumins form stable molten globule states under a range of conditions, with the low pH form being the best characterized. The stability of the molten globule varies among different members of this family, but the origin of the stability difference is not clear. We compare the folding and stability of -subdomain constructs of human and bovine -lactalbumin. Previous studies have demonstrated that the isolated -subdomain of human -lactalbumin folds and forms a molten globule state. The minimum core construct has been defined to include the A, B, and D -helices and the C-terminal 3₁₀ helix. A construct corresponding to the same region of bovine -lactalbumin is much less structured and less stable than the human -lactalbumin construct. Addition of the C-helix to generate a 75-residue bovine construct does not lead to a significant increase in structure or stability. This construct (AB-CD/3₁₀) contains the entire -subdomain of bovine -lactalbumin. Thus molten globule formation in the human protein, but not in the bovine protein, can be rationalized on the basis of a stable -subdomain. Interactions involving more of the protein chain are required to generate a well structured molten globule in the bovine protein. Comparison of AB-CD/3₁₀ to the molten globule formed by the intact protein and to the protein with the 6–120 disulfide reduced indicates that both the -subdomain and the 6–120 disulfide play a role in stabilizing the bovine -lactalbumin molten globule.

Keywords: A-state; -lactalbumin; molten globule; partially folded states; protein folding; protein stability

Abbreviations: AB, a peptide corresponding to residues 1–38 of bovine -lactalbumin • AB-CD/3₁₀, a peptide consisting of residues 1–38 of bovine -lactalbumin cross-linked via the 28–111 disulfide to a peptide consisting of residues 84–120 of bovine -lactalbumin • AB-D/3₁₀, a peptide corresponding to residues 1–38 of bovine -lactalbumin cross-linked via the 28–111 disulfide to a peptide consisting of residues 95–120 of bovine -lactalbumin • ANS, 1-anilinonapthalene-8-sulfonate • BLA_6–120, bovine -lactalbumin with the 6–120 disulfide selectively reduced and blocked by iodoacetamide • CD, circular dichroism • CD/3₁₀, a peptide corresponding to residues 84–120 of bovine -lactalbumin • D/3₁₀, a peptide corresponding to residues 95–120 of bovine -lactalbumin • GnHCl, guanidinium hydrochloride • HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide • HBTU, N-[(1H-benzotriazol-1 yl)(dimethylamino)methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide • HPLC, high-pressure liquid chromatography • MALDI TOF, matrix-assisted laser desorption ionization time of flight mass spectrometry • PAL PEG, 5-(4'mino-methyl-3', 5'-dimethoxyphenoxy) valeryl polyethylene glycol • TFA, tri-fluoroacetic acid

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04977905.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
A growing number of proteins have been shown to populate partially folded states during their equilibrium unfolding. These are commonly referred to as molten globules (Kronman et al. 1965; Dolgikh et al. 1981, 1985; Ohgushi and Wada 1983; Kuwajima 1989; Fink 1995; Ptitsyn 1995; Creighton 1997; Arai and Kuwajima 2000) and are often considered to mimic intermediates populated in kinetic re-folding experiments (Kuwajima 1989; Jennings and Wright 1993; Engelhard and Evans 1995; Fink 1995; Ptitsyn 1995; Creighton 1997; Arai and Kuwajima 2000; Chamberlain and Marqusee 2000). It is therefore important to characterize these equilibrium intermediates in order to better understand the protein folding process. Studies of partially folded states also provide information about the type of interactions needed to specify a unique native state. Interest in molten globules has been heightened by the realization that some proteins aggregate and form amyloid from partially folded states (Kelly 1998; Marmorino and Peilak 1998). The classical definition of the molten globule is a partially folded compact state having a high content of native-like secondary structure but lacking specific tertiary structure (Dolgikh et al. 1981, 1985; Ohgushi and Wada 1983). Some molten globules conform to the original description, but a range of proteins have been shown to form molten globule states that contain specific tertiary interactions (Dolgikh et al. 1981; Ohgushi and Wada 1983; Ptitsyn 1995; Marmorino and Pielak 1998).

The Ca²⁺ binding -lactalbumins form stable molten globule states under a variety of conditions, including low pH, reduction of disulfide bonds, and the absence of Ca²⁺ ions at low ionic strength in the presence of low concentration of urea or guanidinium hydrochloride (GnHCl) (Dolgikh et al. 1981; Ikeguchi et al. 1986; Baum et al. 1989; Kuwajima 1989; Qasba and Kumar 1997; Arai and Kuwajima 2000). The low pH form, commonly called the Astate, is the best characterized. -Lactalbumin is one of the most popular model systems for studies of molten globules. This 123-residue-long protein is divided into two subdomains by a deep cleft (Fig. 1; Acharya et al. 1989, 1991; Pike et al. 1996). The -subdomain comprises residues 1–34 and 86–123, and the -subdomain comprises residues 35–85. The -subdomain is composed of four -helices (denoted A through D) and two short 3₁₀ helices, while the -subdomain is composed of a three-stranded antiparallel -sheet, a series of loops, and a short 3₁₀ helix. There are four intramolecular disulfide bonds in the native protein. One is in the -subdomain, and one bridges the - and -subdomains. The two remaining disulfides are the 28–111 and 6–120 located in the -subdomain. The 6–120 disulfide is hyperreactive and can be selectively reduced without unfolding the protein (Kuwajima et al. 1990).

View larger version (33K): [in this window] [in a new window]   Figure 1. Ribbon diagram of the bovine -lactalbumin structure. The region corresponding to the AB-CD/310 construct is shown in red. Only the 28–111 disulfide bond is shown. The major helices and N-terminus are labeled. The diagram was constructed using the program MOLSCRIPT (Kraulis 1991) and the PDB file (1B90 [PDB] ).

Studies of -lactalbumin have shown that the -subdomain is well structured in the molten globule state, while the -subdomain is significantly less structured but not unfolded (Baum et al. 1989; Kuwajima 1989; Peng and Kim 1994; Schulman et al. 1995, 1997; Creighton 1997; Polverino de Laureto et al. 1999; Troullier et al. 2000). The -subdomain can be nicked or completely removed without eliminating the ability of the -subdomain to form a molten globule-like state, at least for the human protein (Demarest et al. 1999; Peng and Kim 1994; Polverino de Laureto et al. 2002). Previous work performed in our laboratory defined the minimum structured core of human -lactalbumin required to form a stable molten globule. Those studies demonstrated that a construct consisting of the AB helix region cross-linked by the native 28–111 disulfide to the D/3₁₀ helix region forms a stable molten globule state in the absence of the rest of the protein (Demarest et al. 1999, 2001b; Horng et al. 2003). Related peptide constructs derived from hen lysozyme were shown to be largely unstructured. This is expected, since full-length hen lysozyme does not form a stable equilibrium molten globule state in aqueous solution (Demarest et al. 2001a), although it does populate a transient kinetic intermediate with molten globule characteristics. It is not known, however, whether similar peptide constructs from other -lactalbumin proteins form molten globules in the absence of their respective -subdomains.

Here we use this protein dissection approach to compare the properties of the -subdomain of the bovine protein to the human -lactalbumin -subdomain construct. Two constructs were analyzed. The first construct is denoted AB-D/3₁₀ and contains all the residues identified as making up the core of the human -lactalbumin molten globule. The second construct, AB-CD/3₁₀, is larger and includes the C-helix. We also compare the stability of the bovine -subdomain construct to the stability of the molten globule states formed by intact bovine -lactalbumin and by the bovine protein with the 6–120 disulfide bond selectively reduced. These experiments help to elucidate the relative importance of the -subdomain, the 6–120 disulfide, and the -subdomain in stabilizing the bovine -lactalbumin molten globule. These studies help to define which parts of the poly-peptide are important for stabilizing the molten globule state of bovine -lactalbumin, and provide new insight into the origins of the stability differences between the human and bovine molten globules.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Design of bovine -lactalbumin -subdomain constructs
This study is focused on the characterization of two peptide constructs derived from the -subdomain of bovine -lactalbumin and on the comparison with the intact protein and a variant with the 6–120 disulfide reduced. A ribbon diagram of the -lactalbumin structure is shown in Figure 1, and the regions corresponding to the constructs are highlighted. The first construct encompasses the same portion of the polypeptide chain that was included in the human -lactalbumin peptide model, while the second is longer. Both constructs include a peptide fragment corresponding to the A and B helices (residues 1–38) cross-linked via the native 28–111 disulfide bond to fragments that include other portions of the -subdomain. The N-terminal fragment is denoted as AB. The first construct links the AB peptide to a 26-residue peptide, residues 95–120, which includes the D- and C-terminal 3₁₀ helices. The C-terminal fragment is designated the D/3₁₀ peptide. The oxidized 64-residue construct is denoted as the AB-D/3₁₀ peptide and is the bovine analog of the previously studied human -lactalbumin core construct (Demarest et al. 1999). Comparison of the folding properties of this construct to the human construct allows us to determine whether the same minimal structured core applies in the case of bovine -lactalbumin. The second construct has a longer C-terminal fragment, denoted CD/3₁₀. The 30-residue CD/3₁₀ peptide consists of residues 84–120 of bovine -lactalbumin and includes the complete C-helix. The cross-linked construct is denoted AB-CD/3₁₀ and includes all of the helices of the -subdomain. The C-helix is thought to be structured in the bovine -lactalbumin molten globule, and the larger construct allow us to test its role in stabilizing the isolated -subdomain (Alexandrescu et al. 1993; Forge et al. 1999). The Cys residues at positions 6 and 120 were replaced with Ala in order to avoid problems with incorrect disulfide formation. This was also the case for the previously characterized human -lactalbumin construct. Both constructs lack both the -subdomain and the 6–120 disulfide. Thus we compared their apparent stability, as judged by urea denaturation, to that of the molten globules formed by intact bovine -lactalbumin and by bovine -lactalbumin with the 6–120 disulfide reduced and blocked.

Spectroscopic characterization of the isolated -subdomain of bovine -lactalbumin
The constructs were characterized at pH 2.8, chosen to match previous studies of the human -lactalbumin and hen lysozyme constructs, and also at pH 7.0. The AB-D/3₁₀ construct was only partly structured; thus we focused our attention on the longer AB-CD/3₁₀ construct. The shorter AB-D/3₁₀ is partially helical as judged by far UV CD, and the Trp residues are not protected from solvent as judged by fluorescence measurements (Fig. 2). Similar results were obtained at pH 2.8 and pH 7, 25°C. In contrast, the related construct from human -lactalbumin is highly helical, and the helicity is noticeably larger than expected from the normalized sum of the spectra of the reduced AB and D/3₁₀ fragments (Demarest et al. 1999). This later observation shows that interactions between the AB and D/3₁₀ regions induce significant additional structure. No such effect was observed with the bovine -lactalbumin peptides. These observations already reveal key differences between bovine and human -lactalbumin, and demonstrate that the minimum core unit is different for the two proteins.

View larger version (13K): [in this window] [in a new window]   Figure 2. CD spectrum of the oxidized bovine -lactalbumin construct AB-D/310 recorded at pH 2.8 and 25°C. The spectrum is plotted as mdeg vs. wavelength rather than as mean residue ellipticity due to difficulties in obtaining a precession measurement of peptide concentration.

View larger version (20K): [in this window] [in a new window]   Figure 3. CD and fluorescence emission spectra of the oxidized bovine -lactalbumin construct AB-CD/310 recorded at pH 2.8 and 25°C. (A) CD spectrum. (B) Fluorescence emission spectrum. (•) Data collected at pH 2.8 and 25°C, no denaturant; () data collected in the presence of 6 M GnHCl, pH 2.8 and 25°C.

View larger version (22K): [in this window] [in a new window]   Figure 4. Fluorescence emission spectrum of ANS. () 2 µM ANS in the presence of 5 µM AB-CD/310; (•) ANS alone. Measurements were performed at pH 2.8, 25°C.

The bovine construct is considerably less stable than the shorter human construct and less stable than the molten globule state formed by intact bovine -lactalbumin

View larger version (21K): [in this window] [in a new window]   Figure 5. Urea-induced unfolding monitored by CD at 222 nm. (A) The oxidized bovine -lactalbumin construct AB-CD/310. (B) The oxidized human -lactalbumin construct AB-D/310, which consists of residues 1–38 cross-linked to residues 95–120. Data for the human -lactalbumin construct were originally reported by Demarest et al. (1999). (C) The low-pH molten globule state formed by bovine -lactalbumin with the 6–120 disulfide bond selectively reduced and blocked. (D) The low-pH molten globule state formed by bovine -lactalbumin with all disulfides present. Experimental conditions were pH 2.8 and 25°C. Data are plotted as mdeg vs. urea, and the differences in the values at 0 M urea reflect differences in sample concentration.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

 
The experimental results presented here show that there are significant differences in the folding of the isolated -subdomains of bovine -lactalbumin and human -lactalbumin, even though they have similar structure in their native states. The -subdomain of the human protein can form a molten globule state that is highly structured and exhibits a sigmoidal urea unfolding curve. In contrast, the peptide constructs from the -subdomain of bovine -lactalbumin are much less structured. The urea unfolding experiments show that the isolated -subdomain construct of bovine -lactalbumin is also less stable than the -subdomain construct of human -lactalbumin, even though the bovine construct contains the C-helix while the human construct does not. This is an interesting observation, since it shows that the much greater stability of the molten globule of intact human -lactalbumin versus bovine -lactalbumin is likely due in part to an intrinsically more stable -subdomain. These results indicate that at low pH the bovine and human -lactalbumin molten globule states are stabilized by different mechanisms. For human -lactalbumin, formation of the A-state can be rationalized on the basis of a well structured -subdomain that is capable of folding independently. The -subdomain of the bovine protein, in contrast, is less structured, indicating that formation of a stable molten globule requires more of the protein chain. The different behavior of their respective -subdomains indicates that the interactions that stabilize folding intermediates in the -lactalbumins are not rigorously conserved, implying that strict conservation of the detailed structure and energetics of the folding intermediates may not be critical in the evolution of these proteins.

Comparison of the urea unfolding of the AB-CD/3₁₀ construct with the unfolding of the molten globule states formed by bovine -lactalbumin with the 6–120 disulfide reduced and by the intact bovine protein provides clues about the role of the -subdomain and 6–120 disulfide in the A-state. The molten globule formed by the intact protein is much more resistant to urea-induced unfolding, demonstrating that the -subdomain and/or 6–120 disulfide are important. Reduction of the 6–120 disulfide results in a molten globule which is noticeably less resistant to urea denaturation. The molten globule formed by reduced bovine protein appears to still be slightly more resistant to urea-induced unfolding than the AB-D/3₁₀ construct, although the difference in apparent C_m values is only about 0.5 molarurea. Similar results were previously observed when the unfolding of the human -lactalbumin AB-D/3₁₀ construct was compared to the molten globules formed by intact human -lactalbumin and human -lactalbumin with the 6–120 disulfide reduced (Horng et al. 2003). The 6–120 disulfide is clearly very important, but the data suggest that the -subdomain also plays a role in stabilizing the bovine A-state. This is consistent with observations from Fontana and coworkers, who demonstrated that proteolytic fragments that comprise the -subdomain and the C-helix contained some structure, as judged by CD (Polverino de Laureto et al. 2001). In addition, NMR experiments have offered some evidence of structure in the -subdomain in the molten globule state, as have FTIR (Fourier transform infrared) measurements (Alexandrescu et al. 1993; Forge et al. 1999; Troullier et al. 2000).

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Peptide synthesis and purification
Peptide synthesis was performed using an Applied Biosystem Inc. model 433A peptide synthesizer. The amino acid derivatives for the synthesis were purchased from Applied Biosystems and Advanced ChemTech, and the solvents from Fisher Scientific. The AB_1–38, D/3₁₀ and CD/3₁₀ peptides were prepared on a 0.25 mmole scale using standard Fmoc cycles for peptide synthesis. In AB_1–38 and CD/3₁₀, the native Cys residues at positions 6 and 120 were substituted by Ala. Activation steps were carried out using HATU and HBTU as coupling reagents. PAL-PEG resin was used, which gave an amidated C-terminus. The N-terminus of AB_1–38 was not acetylated, but the N-termini of D/3₁₀ and CD/3₁₀ were. The peptides were cleaved from the resin using a cleavage cocktail containing 90% TFA, 3.3% ethanedithiol, 3.3% anisole, and 3.3% thioanisole. The peptides were purified by reverse-phase HPLC with a Vydac C18 column. The composition of the buffers used for HPLC was buffer A 100% H₂O, 0.045% HCl and buffer B 20% H₂O, 80% CH₃CN with 0.045% HCl. The identity of the peptides was confirmed by MALDI mass spectrometry (matrix assisted laser desorption ionization time of flight mass spectrometry).

Formation of peptide dimers
AB-D/3₁₀ was generated by oxidizing a mixture of AB and D/3₁₀ to form the native disulfide. Air oxidation was performed at room temperature for 24 h at pH 8.5. Purified AB_1–38 and D/3₁₀ were mixed in a 1:1 ratio. The concentration of each peptide was determined by absorbance measurements at 280 nm. The extinction coefficients were derived using the Expasy protoparam tools program. Peptides were dissolved in 0.2 M TRIS, and the pH of the mixture was maintained at 8.5. The oxidized AB-D/3₁₀ construct was purified by reverse-phase HPLC after 24 h. The HPLC trace contains three major peaks. The correct peak was identified by MALDI mass spectrometry. The other two peaks corresponded to the homodimers. A similar procedure was used to prepare the oxidized heterodimer AB-CD/3₁₀.

Preparation of selectively reduced bovine -lactalbumin
Bovine -lactalbumin was purchased from Sigma. A modified version of the method of Creighton and coworkers was used to generate the singly reduced form of bovine -lactalbumin as described by Horng et al. (Ewbank and Creighton 1993; Creighton 1997; Demarest et al. 2001b; Horng et al. 2003). Reduction was allowed to proceed for 2 min at 25°C before quenching with io-doacetamide. The three-disulfide protein was separated from un-reduced material by reverse-phase HPLC. The identity of the reduced peptide was verified by electrospray mass spectroscopy.

CD measurements
For CD experiments, a standard buffer of 2 mM phosphate (sodium phosphate, monobasic), 2 mM citrate (citric acid, anhydrous), 2 mM borate (sodium borate) with 10 mM NaCl was used. The pH was adjusted to 2.8 in order to allow direct comparison to earlier studies (Demarest et al. 1999, 2001a). For studies under denaturing conditions, a phosphate buffer with 10 M urea was used. The nonnative buffer was adjusted to pH 2.8. The concentration of the peptide constructs was maintained at 4 µM at pH 2.8 in most of the experiments. An Aviv Model 62A circular dichroism spectrometer was used to perform CD measurements. An averaging time of 3 sec was used for wavelength scans, which were taken with a minimum of five repeats. For far UV CD scans, a cuvette of 1-mm path length and 250 µL sample volume was used. The wavelength scans were performed at 4°C and 25°C to test for any temperature dependence. Concentration dependence experiments were performed by dilution from a concentrated stock solution into buffer. The concentration of the stock solutions was determined by the absorbance at 280 nm. Urea unfolding experiments were monitored by CD at 222 nm at pH 2.8 and 25°C. The concentration of the urea was determined by refractometry.

Fluorescence measurements
An ISA Fluorolog spectrometer was used to perform fluorescence measurements. The concentration of each peptide sample was 4 µM, and measurements were performed at pH 2.8 and 25°C. Trp fluorescence emission measurements were performed using an excitation wavelength of 280 nm. The spectra were recorded over the range 290–500 nm. The ANS (1-anilinonapthalene-8-sulfonate) spectra were recorded over the range 380–650 nm. The excitation wavelength for ANS fluorescence experiments was 370 nm. The ANS concentration was determined using a molar absorption coefficient of 7.8 x 10³ M^–1cm^–1 at 372 nm in methanol. The concentration of the peptide in this experiment was kept at 4 µM, and the ANS was 2 µM in 2 mM phosphate, 2 mM citrate, 2 mM borate, and 10 mM NaCl buffer at pH 2.8. Similar experimental conditions were used at pH 7.0.

	Acknowledgments

 
This work was funded by NIH grant GM 54233 (D.P.R.).

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